Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 191, Issue -, Pages 775-782Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2021.09.132
Keywords
Nitrile hydratase; Sequence-activity relationship; Nicotinamide bio-production
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The study successfully increased the activity and stability of NHase enzyme through sequence analysis and mutagenesis, developing a powerful process for nicotinamide production. The mutant demonstrated significantly enhanced activity, maintained stability, and showed promising potential for industrial production.
The green biocatalyst nitrile hydratase (NHase) is able to bio-transform 3-cyanopyridine into nicotinamide. As the NHase reaction is exothermic, an enzyme with high activity and stability is needed for nicotinamide production. In this study, we used sequence analysis and site-directed mutagenesis to generate a mutant of thermophilic NHase from Pseudonocardia thermophila JCM3095 with substantially enhanced activity and developed a powerful process for nicotinamide bio-production. The specific activity of alpha F126Y/alpha F168Y mutant was successfully increased by 3.98-fold over that of the wild-type enzyme. The half-life of such mutant was longer than 2 h, which was comparable to its parent enzyme. The relative activity of the alpha F126Y/alpha F168Y mutant after treatment with 1 M 3-cyanopyridine and 2 M nicotinamide was 73.2% and 63.7%, respectively, showing minor loss of its original stability. Structural analysis demonstrated that hydrogen bonds at the active site and alpha-beta subunit interface of the NHase contribute to the improved activity and the maintenance of stability. Escherichia coli transformant harboring the mutant NHase was used for nicotinamide bio-production, yielding a nicotinamide productivity of 251.1 g/(L.h), which is higher than the productivity obtained using other NHase-containing strains and transformants. The newly established variant is therefore a promising alternative for the industrial production of nicotinamides.
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